RNA splicing is one of the most important RNA processing steps during viral and eukaryotic gene expression. RNA splicing controls gene expression by removal of non-coding introns and ligation of coding exons before an mRNA could be formed and exported to the cytoplasm for translation. Thus, defining the exon-intron boundary is the first step to involve the accurate recognition of a 5' splice site (5'ss) and a 3' splice site (3' ss) by cellular splicing machinery. My laboratory has focused on understanding how a viral RNA 5' ss or 3' ss is selected and what controls or regulates such a selection. Papillomavirus and Kaposi's sarcoma-associated herpesvirus (KSHV) RNAs have been used in our studies. The two viruses are oncogenic viruses and the pre-mRNAs transcribed from many viral genes, including some viral oncogenes, in those virus genomes undergo extensive RNA splicing regulation. In the past three years, we made some progress in the following areas: 1. RNA 5' cap promotes splicing of a cap-proximal intron in E6E7 pre-mRNA of human papillomavirus (HPV) type 16 in a distance manner. HPV16 and 18 infections via sexual transmission are widely recognized as being responsible for cervical cancer of women in all parts of the world. High prevalence and incidence of cervical HPV infection have been observed among HIV-positive women and cervical cancer has been the most common malignancy among the women with AIDS in both Europe and the USA. Two viral oncoproteins E6 and E7 of HPVs are involved in carcinogenesis of cervical cancer and respectively, inactivate cellular tumor suppression proteins p53 and pRB. HPV16 E6 and E7 are transcribed, from the same promoter p97, as a bicistronic RNA that has an intron in the E6 coding region with one 5' ss and two alternative 3' ss, producing E6*I and E6*II. The E6E7 mRNA with this intron unspliced expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA can be spliced in vitro only when capped. The cap-dependent splicing of the E6E7 pre-mRNA is extremely efficient in cervical cancer-derived cells, producing mostly E6*I, but inefficient in a common retrovirus expression vector pLXSN16E6E7-transfected cells, due to the large size of its exon 1. Further studies show that an efficient splicing of the E6E7 pre-mRNA depends on distance of a cap-proximal intron from the RNA 5' cap, with an optimal distance less than 304 nts. The same was true for splicing of human b-globin RNA. Splicing of the E6E7 pre-mRNA provides more E7 RNA templates and promotes E7 expression. Data indicate that the distance between RNA 5' cap and cap-proximal intron is rate-limiting for cap-dependent RNA splicing. 2. By limiting HPV16 E6E7 RNA splicing based on our finding, we have identified three novel nuclear localization signals (sequences) (NLS) responsible for accumulation of HPV16 E6 in cell nucleus. Low-risk, non-oncogenic HPV E6s lacking those NLS sequences are cytoplasmic proteins and can be converted into a nuclear protein by insertion of HPV16 E6 NLS sequences to their corresponding regions. These findings may have a great impact on our understanding how a high-risk, oncogenic HPV causes cervical cancer. 3. Regulation of alternative 3' ss selection by cellular splicing factors has been approached by using a bovine papillomavirus type 1 (BPV-1) late RNA transcripts since BPV-1 late transcripts has been used as our model in the past to understand the mechanisms that control the alternative 3' ss usage. Two cellular splicing factors, SR protein ASF/SF2 and a cold-shock protein YB-1, have been found involving in vivo selection of the two alternative 3' splice sites. ASF/SF2 prefers a proximal over a distal 3' splice site, whereas the reverse is true for the YB-1 that prefers a distal over a proximal 3' splice site. 4. Splicing profiling of viral RNA transcripts has been approached by studies on KSHV gene expression. KSHV ORF50, K8 and K8.1 genes are our focus. Three genes are positioned side by side in the virus genome, but belong to three different categories of viral lytic genes. ORF50 is an immediate-early gene encoding an essential transcription activator Rta that is absolutely required to initiate virus lytic infection. K8 is an early gene encoding a DNA replication-associated protein K-bZIP. K8.1 is a late gene responsible for production of a viral envelope glycoprotein for virus maturation and assembly. We have extensively analyzed all three transcripts of ORF50, K8 and K8.1 and found that they all overlap each other in order to share a common poly (A) site downstream of the K8.1 coding region. Interestingly, K8 exon 3 (ORF50 exon 4) has been found bearing two additional 5' ss which could be used for alternative RNA splicing during viral gene expression. By doing so, transcription of these three genes produces more than 19 RNA species. Some of these transcripts are real messages and others are splicing intermediates. This observation has lead us to demonstrate that a K8b RNA is a splicing intermediate and precursor of K8a responsible for encoding k-bZIP. In addition, we have mapped for the first time that transcription of the K8.1 can be activated at a transcription start site nt 75901 in the virus genome and a 24-bp K8.1 promoter containing a 12-bp, TATA-like sequences upstream of the start site is responsible for the transcription activation which is sensitive to inhibition by viral DNA polymerase inhibitors, PAA and GCV.